Preclinical Evaluation of Combination Therapy of Rolipram and Minocycline for Arresting Secondary Injury Cascade After Traumatic Brain Injury

项目摘要 对于大多数创伤性脑损伤（TBI），在撞击发生的那一刻， 损害然而，在撞击后的几分钟内，大脑中的内源性反应会放大并延续损伤 歧管这种级联反应被称为继发性损伤，人们认为这一过程会导致 长期的神经问题我们的目标是迅速减轻一些促成因素， 二次损伤，以减少长期损伤和神经损伤。我们将使用一种独特的组合 包括使用最近开发的共聚物，聚乙烯， （丙交酯-共-乙交酯）-接枝-聚乙烯亚胺（PgP）作为纳米载体。形态学，生理学，化学， 并在术后一个月内的几个时间点记录继发性损伤的行为标志物。 在小鼠中进行实验性TBI，以确定药物组合是否在较早的时间点减少继发性损伤 如果它在受伤后一个月内保持这种减少，我们将使用一种独特的 多模式系统，以监测影响，包括一个新开发的微丝生物传感器谷氨酸（GLU） 和γ-氨基丁酸（GABA）与改良的微棱镜植入物，以关联GLU和 GABA信号和持续的继发性细胞损伤，通过观察相同的细胞随着时间的推移。我们高度 敏感的和选择性的GLU和GABA微生物传感器将被用来评估治疗对 在真实的中，通过测量皮质中的细胞外浓度和释放动力学来减少兴奋性毒性， 时间，在TBI后5个时间点与伤前基线相比。在同一时间点，高分辨率多光子 通过永久植入的改良棱镜进行显微镜检查，以便于垂直观察皮质 层3-6，以监测树突和轴突上的静脉曲张（sxD）的发展，轴突起伏， 收缩球和轴突的消失。此外，通过查看相同的细胞随时间的变化，我们将 比较这些药物对表现出损伤的细胞上的树突和轴突静脉曲张的解决能力， 之前的影像学检查治疗效果也将使用标准的运动行为测试进行评估。 协调，探索和记忆，并通过使用已建立的测定和抗体染色和细胞因子 二次损伤的分子生物标志物的测定。通过比较我们的新组合的结果， 光学成像和实时谷氨酸和GABA信号传导， 和分子生物标志物测试在匹配的时间点，我们将证明这种纵向的，光学- 生物传感器系统来量化二次损伤及其解决方案。值得注意的是，这种纵向方法将 需要更少的动物，因为每只动物都作为自己的对照，减少了变异性， 多个时间点。此外，光学生物传感器数据从受伤，车辆治疗的小鼠将提供新的 更好地理解TBI后继发性损伤和神经变性的级联反应。 此外，这种新的光学生物传感器系统将成为评估其他药物和 优化治疗窗口。

PROJECT SUMMARY For most traumatic brain injuries (TBI), the moment that an impact occurs, there is surprisingly little apparent damage. Yet, within minutes of impact, endogenous responses in the brain amplify and perpetuate the damage manifold. This cascade of responses is called secondary injury, and it is thought that this process can lead to long-term neurological problems. Our goal is to quickly mitigate some of the contributing elements of secondary injury to reduce long-term damage and neurological impairments. We will use a unique combination of drugs, including noninvasive delivery of one drug to the brain using a recently-developed co-polymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) as a nanocarrier. Morphological, physiological, chemical, and behavioral markers of secondary injury will be recorded at several time points up to one month after experimental TBI in mice to determine if the drug combination reduces secondary injury at an earlier time point than either drug alone, and if it maintains this reduction through one month after injury. We will use a unique multimodal system to monitor effects comprised of a newly developed microwire biosensor for glutamate (GLU) and γ-aminobutyric acid (GABA) with a modified micro-prism implant to correlate the dynamics of GLU and GABA signaling and ongoing secondary cellular damage by observing the same cells over time. Our highly sensitive and selective GLU and GABA microbiosensor will be used to evaluate the effect of treatment on reducing excitotoxicity by measuring extracellular concentrations and release dynamics in the cortex, in real time, at 5 time points after TBI versus preinjury baseline. At the same time points, high-resolution multiphoton microscopy through a permanently-implanted, modified prism will be used to facilitate a vertical view of cortical layers 3-6, to monitor development of varicosities (swellings) on dendrites and axons, axonal undulations and retraction bulbs, and the disappearance of axons. Furthermore, by viewing the same cells over time, we will compare the ability of the drugs to resolve dendritic and axonal varicosities on cells exhibiting damage in previous imaging sessions. Therapeutic effects will also be evaluated using standard behavioral tests for motor coordination, exploration, and memory, and by using established assays and antibody staining and cytokine assays for molecular biomarkers of secondary injury. By comparing the results from our novel combination of optical imaging and real-time glutamate and GABA signaling with the results from well-established behavioral and molecular biomarker tests at matched time points, we will demonstrate the utility of this longitudinal, optical- biosensor system to quantify secondary damage and its resolution. Notably, this longitudinal approach will require fewer animals because each animal serves as its own control, reducing variability, and each is used at multiple time points. In addition, optical-biosensor data from injured, vehicle-treated mice will provide new insights for better understanding the cascade of secondary injury and neural degeneration after TBI. Furthermore, this new optical-biosensor system will become a valuable tool for evaluating other drugs and for optimizing therapeutic windows.